Subclinical endotoxemia (low levels of circulating bacterial endotoxin) has been observed in patients suffering from chronic inflammatory diseases such as atherosclerosis, diabetes, and obesity. However, the link between this condition and chronic inflammation is poorly understood. Previous work from our lab has shown that chronic exposure to super-low doses of bacterial endotoxin (LPS) aggravates atherosclerosis resulting in increased plaque size and instability in a macrophage-dependent manner in a mouse model of atherosclerosis. Further, we showed that super-low dose LPS (SLD-LPS) treatment was able to inhibit lysosomal fusion in immortalized macrophages. However, this was done under more acute treatment conditions. The aim of this project was to examine the molecular mechanisms by which chronic SLD-LPS may polarize macrophages to a non-resolving pro-inflammatory state consistent with chronic inflammation. This was carried out in two projects, the first a more broad phenotypic paper showing the disruption in homeostasis by chronic SLD-LPS in immortalized macrophages, while the second uses primary bone marrow-derived mouse macrophages to identify specific molecular signaling pathways used by chronic SLD-LPS. Here we show that chronic SLD-LPS led to the novel upregulation of pro-inflammatory mediators p62 and ccl2 with simultaneous downregulation of homeostatic mediators Nrf2 and slc40a1 in immortalized wild-type mouse macrophages. Further we showed this effect was reversed using the homeostatic restorative agent sodium phenylbutyrate (4-PBA), a newly reported activity for this reagent in mouse macrophages. This indicated that a disruption in homeostasis, possibly involving autophagy, may be responsible for the non-resolving pro-inflammatory polarization of macrophages. Therefore, in our second project, we further explored the effect of chronic SLD-LPS treatment on the homeostatic arm of the response by focusing on the Nrf2 inhibitor Keap1. Here we show that chronic SLD-LPS results in an accumulation of Keap1 in mouse bone marrow-derived macrophages, an effect specific to chronic SLD-LPS, as high doses of LPS failed to induce Keap1. We suggest that this effect may be related to a disruption in lysosomal fusion as evidenced by accumulation of autophagy flux markers MLKL and p62. Further, we show that these effects are dependent on the non-traditional TLR4 adaptor TRAM, suggesting an alternative dose-dependent signaling pathway for LPS. Together this work identifies novel signaling mechanisms involved in non-resolving pro-inflammatory polarization of murine macrophages, providing new insight behind how chronic super-low dose LPS exposure may lead to chronic inflammation. / Doctor of Philosophy / Inflammation is the body's natural response to injury or insult and can be beneficial in certain contexts such as pathogen clearance. However, left un-checked, chronic inflammation can exacerbate or even lead to disease pathology, such as is the case with modern diseases such as atherosclerosis, obesity, diabetes, etc. Despite the high prevalence of these diseases, effective treatments and therapies are still lacking. Recently it was discovered that many patients suffering from chronic inflammatory diseases had low levels bacterial endotoxin (LPS) in their circulation, a condition referred to as subclinical endotoxemia. However, possible links between this condition and chronic inflammatory disease remain poorly understood. Using a mouse model of atherosclerosis, previous research from our lab showed that persistent exposure to super-low doses of bacterial endotoxin (similar to those observed in humans) lead to aggravated atherosclerosis with both increased plaque size and instability. Further, we showed that this effect was primarily mediated by pro-inflammatory polarized immune cells called macrophages, but the molecular mechanism behind this polarization is still unclear. Further research into these molecular mechanisms may provide better targets for the development of future chronic inflammatory disease treatments. Here using a combination of mouse cell line and primary cell cultures, we discuss how chronic exposure to super-low doses of bacterial endotoxin leads to the chronic non-resolving pro-inflammatory polarization of macrophage immune cells, with particular emphasis on the distinct molecular signaling mechanisms induced by chronic super-low dose LPS.

Orientador: Vânia Niéto Brito de Souza / Resumo: A infecção pelo Mycobacterium leprae (ML) estimula um processo de desdiferenciação e proliferação das células de Schwann (SCs) que pode contribuir para a disseminação do bacilo. Os macrófagos (MOs) são células efetoras da resposta imune que promovem a eliminação de patógenos, entretanto, na hanseníase são colonizados pelo ML. Sabe-se que os MOs podem apresentar uma polarização funcional na qual os MOs M1 apresentam características pró-inflamatórias e microbicidas enquanto os MOs M2 atuam na reparação tecidual e possuem perfil anti-inflamatório. SCs infectadas pelo ML produzem mediadores capazes de interferir com a função dos MOs aumentando sua sobrevida e promovendo sua migração. Embora diferentes programas funcionais tenham sido observados em MOs de pacientes com formas polares da hanseníase a influência de SCs nesse processo não é sabida. Neste estudo avaliamos se SCs infectadas pelo ML podem interferir na polarização de MOs murinos derivados de medula óssea. Para tanto, culturas primárias de SCs murinas foram infectadas experimentalmente com bacilos viáveis e cocultivadas com MOs. Nossos resultados indicam que a produção de óxido nítrico foi baixa nas culturas de MOs após a infecção com o bacilo, mas mostrou-se aumentada nas coculturas de MOs e SCs infectadas pelo ML. A infecção com ML não induziu produção significante das citocinas IL-6, IL-10 e TNF em culturas de MOs e SCs, entretanto, a interação entre MOs e SCs infectadas com o bacilo resultou em aumento na produção de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mycobacterium leprae (ML) infection stimulates dedifferentiation and proliferation of Schwann cells (SCs) that may contribute to the spread of the bacillus. Macrophages (MOs) are effector cells of the immune response that promote the elimination of pathogens, however, in leprosy they are colonized by ML. It is known that MOs can present a functional polarization in which M1 MOs show pro-inflammatory and microbicidal activities while M2 MOs act in tissue repair presenting an anti-inflammatory profile. SCs infected by ML produce mediators able to interfere with MOs function, increasing their survival and promoting their migration. Although different functional programs have been observed in MOs from patients with polar forms of leprosy, the influence of SCs in this process is not known. In this study we evaluated whether SCs infected with ML could interfere in the polarization of murine MOs derived from bone marrow. For this purpose, primary cultures of murine SCs were experimentally infected with viable bacilli and co-cultivated with MOs. Our results indicate that nitric oxide production was low in cultures of MOs after infection with the bacillus, but it was increased in the co-cultures of MOs and ML-infected SCs. The infection with ML did not induce significant production of IL-10, TNF and IL-6 in cultures of MOs and SCs, however, the interaction between MOs and ML infected-SCs resulted in increased production of cytokines, mainly IL-10, inducing a decrease in the TNF/IL-10 ... (Complete abstract click electronic access below) / Mestre

La pemphigoïde bulleuse (PB) est la plus fréquente et la plus grave des dermatoses bulleuses auto-immunes caractérisée par une cascade inflammatoire impliquant plusieurs cytokines et cellules inflammatoires, avec la libération de protéases (MMP-9, élastase) conduisant à la formation de la bulle. L’objectif de cette étude est de caractériser cette cascade inflammatoire en analysant la contribution de la chimiokine CXCL10 et des cellules inflammatoires comme les macrophages dans le mécanisme physiopathologique associé à la maladie. Nos résultats ont montré que CXCL10 est présent à des taux importants dans le sérum des patients et dans le liquide de bulle, de plus cette chimiokine reste fortement présente dans le sérum des patients qui rechutent. D’autre part nous avons montré pour la première fois que l’orientation des macrophages dans la PB est contrôlée dans un premier temps par les sérums de patients, qui préactivent les macrophages dérivés de monocytes, puis par le liquide de bulle qui différencient les macrophages vers une polarisation M2. Nous avons également montré que seulement les neutrophiles et les monocytes isolés de patients atteints de PB, mais pas leur lymphocytes, répondaient à une stimulation par CXCL10 par une augmentation de leur sécrétion en MMP-9 via l’activation des voies de signalisation ERK1/2, P38 et PI3K. Cette expression de la MMP-9 a été associée à une polarisation de type M2 dans les macrophages issus de monocytes de patients. Enfin, nous avons montré l’importance des activateurs sélectifs des récepteurs des glucocorticoïdes, tels que le composé A, qui était aussi efficace que le traitement avec la méthylprénisolone dans l'inhibition de la MMP-9, mais avec des effets distincts sur l'expression des marqueurs phénotypiques de macrophages. / Bullous pemphigoid (BP) is the most common and serious autoimmune bullous dermatosis characterized by inflammatory cascade involving many cytokines and inflammatory cells, with the release of proteases (MMP-9, HLE) leading to the formation of the blister. The objective of this study is to characterize the inflammatory cascade by analyzing the contribution of the CXCL10 chemokine and inflammatory cells such as macrophages in the pathophysiology associated with the disease. Our results showed that CXCL10 is present at significant levels in the serum of patients and in the blister fluid, this chemokine remains strongly present in the serum of patients who relapse. On the other hand we have shown for the first time that macrophages polarization in BP is controlled by the sera of patients, which activate macrophages derived from monocytes, and then blister fluid differentiate macrophages in an M2 polarization. We also showed that only neutrophils and monocytes isolated from patients, but not the lymphocytes responding to stimulation by CXCL10 by an increase in secretion of MMP-9 via the activation of signaling pathways ERK1 / 2, P38 and PI3K. This expression of MMP-9 has been associated with an M2 macrophages polarization derived from monocytes of patients. Finally, we have demonstrated the importance of selective glucocorticoid receptor activators, such as Compound A, which was as effective as treatment with methylprednisolone in inhibiting MMP-9, but with distinct effects on the expression of phenotypic markers of macrophages.

Notre équipe étudie les HSP, et notamment HSP110. Les HSP sont des chaperons impliqués dans le repliement des protéines nouvellement synthétisées et dénaturées. Les HSP sont surexprimées lors des stress et participent à la survie des cellules par leurs propriétés anti-apoptotiques et anti-agrégations. HSP110 est surexprimée dans le cancer colorectal et est associée à un mauvais pronostic. L’expression d’un mutant d’HSP110, nommé HSP110DE9, a été mise en évidence dans les cancers colorectaux de type MSI. Celui-ci y agit comme un dominant négatif, en se liant à HSP110 et en inhibant ses fonctions. Son expression sensibilise les cellules cancéreuses à la chimiothérapie et est associée à un bon pronostic chez les patients.Je me suis tout d’abord intéressé au rôle d’HSP110 dans la régulation de la voie oncogénique STAT3. Son activation est en effet associée à un mauvais pronostic, par l’induction de gènes impliqués dans la prolifération et la survie. La protéine HSP110 favorise la prolifération des cellules colorectales cancéreuses à travers cette voie. HSP110DE9 en revanche l’inhibe. Je me suis ensuite intéressé au rôle d’HSP110 sur la polarisation des macrophages dans le cancer colorectal. Celle-ci peut être sécrétée par les cellules cancéreuses et induit une polarisation pro-tumorale des macrophages. HSP110DE9, en bloquant la sécrétion d’HSP110, conduit en revanche à une polarisation pro-inflammatoire. L’effet d’HSP110 sur la polarisation implique le récepteur TLR4.L’ensemble de ces résultats montrent le rôle d’HSP110 dans la progression tumorale. HSP110 apparaît comme une cible thérapeutique dans le traitement du cancer colorectal. / Our team studies HSPs, including HSP110. HSPs are chaperones involved in the folding of newly synthesized and denatured proteins. HSPs are overexpressed under stress conditions and are involved in cell survival thanks to their anti-apoptotic and anti-aggregation functions. HSP110 is overexpressed in colorectal cancer and is associated with a poor prognosis. The expression of a mutant HSP110, named HSP110DE9, has been shown in MSI colorectal cancer. This one was shown to act there as a dominant negative, by binding HSP110 and inhibiting its functions. Its expression sensitizes cancer cells to chemotherapy and is associated with a better prognosis for patients.I was first interested in HSP110 role in regulating the oncogenic STAT3 pathway. Its activation is associated with a poor prognosis, as it induces the transcription of genes involved in proliferation and survival. HSP110 favors colorectal cancer cell proliferation through this pathway. Conversely, HSP110DE9 inhibited it.I then focused on the role of HSP110 on macrophage polarization in colorectal cancer. HSP110 can be secreted by cancer cells and induces a pro-tumoral macrophage polarization. In contrast, HSP110DE9, by inhibiting HSP110 release, leads to a pro-inflammatory polarization. HSP110 effect on macrophage polarization involve the TLR4 receptor.All these results show HSP110 role in tumor progression. HSP110 appear as a therapeutic target in the treatment of colorectal cancer.

Choline is necessary for the synthesis of phosphatidylcholine (PC), the predominant phospholipid species and an important lipid intermediate. Macrophages, critical mediators of innate immunity, have been implicated in lipid dysregulation associated with metabolic disease. Despite the importance of choline in lipid metabolism, few studies have investigated the relationship between choline metabolism and inflammation. My research revealed that macrophage polarization increased choline metabolism and the expression of the choline transporter CTL1. In addition, choline deficient macrophages showed altered cytokine secretion, suggesting choline metabolism may play an important role in regulating the immune response. This study also describes the generation of a novel CTL1-/- mouse, which showed decreased choline uptake and incorporation into lipids. As an in vivo model for choline deficiency, CTL1-/- mice represent an important model for the future study of choline metabolism. Altogether, these findings suggest an important relationship exists between choline metabolism and inflammation.

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Muscular dystrophies are a heterogeneous group of genetic diseases that involve mutations in genes leading to progressive muscular weakness. Limb-Girdle Muscular Dystrophy 2B (LGMD2B) is a subset of muscular dystrophy caused by mutations in the DYSF gene, which encodes for dysferlin protein and has an incidence of 1/100,000-1/200,000 people, or 1/300 people of Libyan Jewish descent. Since there is no effective treatment that can cure or reverse effects of LGMD2B once diagnosed, our goal is to investigate and develop a protein therapy that mitigates effects of this disease in patients. Galectin-1 (Gal-1) is a small, soluble 14.5 kDa protein with a carbohydrate recognition domain capable of stabilizing the sarcolemma. The exact role that Gal-1 plays in myogenic cells is not fully understood, however, it is known that Gal-1 possesses anti-inflammatory properties and increases the terminal differentiation of committed myogenic cells. Our hypothesis is that Gal-1 treatment increases myogenic potential, improves membrane repair capability, and modulates the immune response in models of LGMD2B by stabilizing muscle integrity, leading to decreased disease manifestation. To test this hypothesis and assess the effect of Gal-1 treatment on myogenesis, anti-inflammatory modulation, and membrane repair, we designed, produced, and purified recombinant human galectin-1 (rHsGal-1) to be used in LGMD2B models. Our in vitro results indicate that after 2-3 days of treatment with 0.11μM rHsGal-1, A/J-/- myotubes enhance expression of myogenic late markers and increase in size and alignment. Additionally, after short-term treatment, rHsGal-1 improves membrane repair capability in a Ca2+ independent manner through an activated carbohydrate recognition domain (CRD) in in vitro and in vivo models of LGMD2B. We give evidence that rHsGal-1 upregulates anti-inflammatory cytokines, increases functional activity, and modulates the canonical NF-κB inflammatory pathway in dysferlin-deficient models by decreasing expression of TAK-1 and the p65 and p50 subunits in vitro and short-term in vivo treatment. Similar effects of the rHsGal-1 treatment were observed in patient-derived dysferlin-deficient human myotubes. Exploratory results show a potential decrease in muscle fat deposition in Bla/J mice. Furthermore, Gal-1 contributes to immune modulation by helping to initiate muscle regeneration by shifting M2 macrophage polarization. Together, our novel discoveries provide direct evidence that Gal-1 is a promising candidate to treat LGMD2B disease pathologies by improving expression of late-stage myogenic markers, improving membrane repair in vitro and short-term in vivo studies, promoting muscle regeneration through immune modulation, and reducing canonical NF-κB inflammation.

Macrophages (MF) are known to exhibit distinct responses to viral and bacterial infection, but how they react when exposed to the pathogens in succession is less well understood. Accordingly, we determined the effect of a rubella virus (RV)-induced infection followed by an LPS-induced challenge on cytokine production, signal transduction and metabolic pathways in human GM (M1-like)- and M (M2-like)-MF. We found that infection of both subsets with RV resulted in a low TNF-a and a high interferon (IFN, type I and type III) release whereby M-MF produced far more IFNs than GM-MF. Thus, TNF-a production in contrast to IFN production is not a dominant feature of RV infection in these cells. Upon addition of LPS to RV-infected MF compared to the addition of LPS to the uninfected cells the TNF-a response only slightly increased, whereas the IFN-response of both subtypes was greatly enhanced. The subset specific cytokine expression pattern remained unchanged under these assay conditions. The priming effect of RV was also observed when replacing RV by IFN-b one putative priming stimulus induced by RV. Small amounts of IFN-b were sufficient for phosphorylation of Stat1 and to induce IFN-production in response to LPS. Analysis of signal transduction pathways activated by successive exposure of MF to RV and LPS revealed an increased phosphorylation of NFkB (MMF), but different to uninfected MF a reduced phosphorylation of ERK1/2 (both subtypes). Furthermore, metabolic pathways were affected; the LPS-induced increase in glycolysis was dampened in both subtypes after RV infection. In conclusion, we show that RV infection and exogenously added IFN-b can prime MF to produce high amounts of IFNs in response to LPS and that changes in glycolysis and signal transduction are associated with the priming effect. These findings will help to understand to what extent MF defense to viral infection is modulated by a following exposure to a bacterial infection.

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